Aralkylation of guanosine with para-substituted styrene oxides

Article abstract of DOI:10.1021/tx970126i

To probe mechanisms of nucleoside aralkylation, product distributions and product stereochemistries were determined in reactions of optically active p-methyl- and p-bromostyrene oxide with guanosine. The proportion of 7-, N²-, and O⁶

Full text of DOI:10.1021/tx970126i

44
Chem. Res. Toxicol. 1998, 11, 44-53
Ar a lk yla tion of Gu a n osin e w ith P a r a -Su bstitu ted
Styr en e Oxid es
Thomas Barlow* and Anthony Dipple
Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick
Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702
Received J uly 21, 1997X
To probe mechanisms of nucleoside aralkylation, product distributions and product stereo-
chemistries were determined in reactions of optically active p-methyl- and p-bromostyrene
oxide with guanosine. The proportion of 7-, N2-, and O6-substituted guanosine products was
∼0.32:0.62:0.06 in neutral, aqueous reactions with the (R)-p-methylstyrene oxide and ∼0.85:
0.09:0.04 in reactions with the (R)-p-bromostyrene oxide. The exocyclic positions opened the
epoxide at the R-carbon. Epoxide ring opening by the nitrogen at the 7-position showed little
preference for the R- or â-carbons in reactions with p-methylstyrene oxide. However, the
p-bromostyrene oxide favored reaction at the â-carbon almost 4-fold over reaction at the
R-carbon. Almost total inversion of stereochemistry was found to occur in reactions at the
7-position. In contrast, the ratio of inversion to retention of configuration in N2- and
O6-substituted products was ∼2:1 and ∼1:1 for reactions with the p-methylstyrene oxide and
∼6:1 and ∼3:1 for reactions with p-bromostyrene oxide, respectively. These experiments suggest
that an SN2 mechanism is in effect with reactions at the 7-position, whereas substrates of an
increasingly ionic nature are involved in reactions at the N2- and O6-positions, respectively.
also reacted in aqueous solution with all three guanosine
centers (23, 24). The 7-position opened the oxirane ring
at both the R- and â-carbons, and N2- and O6-substituted
products were formed at the R-carbon (18, 23, 24).
Optically active forms of the epoxide have been used to
provide additional mechanistic information from the
stereochemical dimension (18).
In the present study, mechanistic investigations were
extended by using optically active p-methyl- and p-
bromostyrene oxide to further explore the factors control-
ling aralkylation of guanosine.
In tr od u ction
Chemical carcinogens initiate the carcinogenic process
by binding to constituents of DNA (1). Many of these
carcinogens are not reactive themselves but require
metabolic activation to become DNA-reactive agents (2).
These latter ultimate carcinogens are often formed by
epoxidation of unsaturated bonds, and it is the resultant
oxidation products that react with DNA to initiate
mutagenic and carcinogenic processes (2). Although
many ultimate carcinogens share the same reactive
epoxide moiety, the spectrum of DNA adducts formed by
these compounds is varied. Simple alkyl epoxides such
as ethylene oxide (3) and propylene oxide (4) predomi-
nantly target the 7-position of guanine residues (5),
whereas alkylnitrosoureas react at the O6-position in
addition to the 7-position (1), and the dihydrodiol ep-
oxides of some polycyclic aromatic hydrocarbons such as
benzo[a]pyrene (6-8) and 5-methylchrysene (9-11) react
almost exclusively at the exocyclic amino group of
guanine in DNA. This differing site selectivity has
prompted several studies exploring the underlying mech-
anisms involved (12-19).
The sites on guanosine that are modified most exten-
sively by electrophilic carcinogens are the 7-, N2-, and
O6-positions (15). Studies using benzylating agents that
modify all three positions indicated that reactions at the
ring nitrogen follow a bimolecular displacement mecha-
nism whereas modification of the exocyclic groups re-
quires some degree of substrate ionization for reaction
to occur (14-17). These indications were supported by
later studies which probed reactions at these sites with
styrene oxide (18). Styrene oxide, the ultimate carcino-
gen formed from metabolic oxidation of styrene (20-22),
Exp er im en ta l Section
Ca u tion : The para-substituted styrene oxides synthesized
may be potentially mutagenic and/or carcinogenic and should
be handled with care.
Ch em ica ls. All chemicals were obtained from Aldrich
Chemical Co. (Milwaukee, WI) with the exception of hydroqui-
nidine 1,4-phthalazinediyl diether [(DHQD)2PHAL],1 hydroqui-
nine 1,4-phthalazinediyl ether [(DHQ)2PHAL], and potassium
osmate dihydrate which were obtained from Fluka (Ronkonko-
ma, NY) and generally labeled [14C]guanosine (specific radio-
activity 509 mCi/mmol) which was obtained from Amersham
Searle (Arlington Heights, IL). 2-Chloro-6-hydroxypurine was
prepared by the method of Shapiro et al. (25). TLC was carried
out on 60 F 254 silica gel on aluminum plates obtained from
Aldrich Chemical Co. (Milwaukee, WI) using 2:8 ethyl acetate/
hexane as a solvent system. Wet flash chromatography was
carried out using silica gel 60 obtained from Aldrich Chemical
Co. Ecoscint A scintillant was obtained from National Diag-
nostics (Atlanta, GA).
In str u m en ta tion . HPLC was carried out on a Hewlett-
Packard model 1090 high-pressure liquid chromatography
instrument equipped with a diode array detector and a YMC J ′
1
Abbreviations: CE, capillary electrophoresis; (DHQD)2PHAL, hy-
* Corresponding author.
droquinidine 1,4-phthalazinediyl diether; (DHQ)2PHAL, hydroquinine
1,4-phthalazinediyl diether; ee, enantiomeric excess.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
S0893-228x(97)00126-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/19/1998

Mechanisms of Guanosine Aralkylation
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 45
sphere ODS-M80 250- × 4.6-mm column (Wilmington, NC).
Chiral separations were achieved by capillary electrophoresis
on a Beckman instrument equipped with an XAA 47- × 50-mm
column using 10 mM phosphate adjusted to pH 2.3 by triethyl-
amine and 5 mM chiral selector sulfyl butyl ether (IV)-â-
cyclodextrin [â-CD-SBE (IV)] as the mobile phase. Ultraviolet
absorption spectra were recorded with a Milton Roy Spectronic
3000 diode array spectrophotometer. Circular dichroism spectra
were measured on a J asco model J 500A spectropolarimeter
equipped with a data-processing system for signal averaging.
1H NMR spectra were obtained using either a Varian XL-200
instrument or a Varian-500S instrument. COSY homonuclear
2D spectra were obtained using the Varian-500S instrument.
Samples were dissolved in CDCl3 or DMSO-d6 with tetrameth-
ylsilane as an internal standard. Positive ion (+ve) electron
CH3); +ve EI-MS m/z 152.083 50 ([M]+; calcd for C9H12O2,
152.083 73).
(S)-(p-Br om op h en yl)-1,2-eth a n ed iol: 6.13 g, 74%; Rf 0.21;
1H NMR (CDCl3) δ 7.50 (d, 2, Ar, J ) 8.5 Hz), 7.26 (d, 2, Ar, J
) 8.3 Hz), 4.80 (dd, 1, R-CH, J R,â1,â2 ) 3.6, 4.4 Hz), 3.80-3.57
(m, 2, â-CH1,2), 2.21 (br s, 2, OH); +ve EI-MS m/z 217.978 16
([M]+; calcd for C8H9O281Br, 217.976 54), 215.979 67 ([M]+; calcd
for C8H9O279Br 215.978 59).
Syn th esis of P ar a-Su bstitu ted P h en yleth an e 1-Hydr oxy-
2-tosyla tes. Para-substituted phenyl-1,2-ethanediol (6.9 mmol,
1 equiv) was coevaporated with anhydrous pyridine (3 × 10 mL)
and then redissolved in anhydrous pyridine (20 mL). The
solution was cooled to 0 °C before the dropwise addition of tosyl
chloride (1.45 g, 7.6 mmol, 1.1 equiv) solution in anhydrous
pyridine (10 mL) with vigorous stirring. The mixture was
stirred for 16 h, then coevaporated with toluene, and concen-
trated in vacuo. The resulting gum was dissolved in ethyl
acetate (50 mL) and washed with water (50 mL). The product
was extracted into the organic phase with ethyl acetate (3 × 50
mL), and the combined organic phase was dried over magnesium
sulfate, filtered, and concentrated in vacuo before purification
by wet flash column chromatography (silica, eluted with a
gradient of 0-40% ethyl acetate in hexane) to yield the product
as a white solid (yield 81-91%).
impact (EI) mass spectra (MS) were obtained with
a VG
Analytical 72-50 instrument, and +ve fast atom bombardment
(FAB) MS were obtained with a reverse-geometry VG Micromass
ZAB-2F spectrometer interfaced to a VG 203S data system using
glycerol as the FAB matrix. For bromo compounds, the ratio
of abundance of 79Br/81Br isotopes was in the range of 1.07-
0.91.
Syn th esis of (()-P a r a -Su bstitu ted P h en yl-1,2-eth a n e-
d iols. Potassium ferricyanide (37.44 g, 114 mmol, 3 equiv) and
potassium carbonate (15.72 g, 114 mmol, 3 equiv) were dissolved
in 50% (v/v) tert-butyl alcohol (380 mL), and potassium osmate
dihydrate (27.8 mg, 75 mmol, 0.002 equiv) was added. The
biphasic mixture was cooled to 0 °C, para-substituted styrene
(38.2 mmol, 1 equiv) was added, and the mixture was stirred
for 8 h at 0 °C and for an additional 16 h at room temperature.
Sodium sulfite (57.3 g) and ethyl acetate (200 mL) were added,
and the mixture was stirred for 30 min. The product was
extracted with ethyl acetate (3 × 100 mL), and the combined
organic phase was dried over magnesium sulfate and concen-
trated in vacuo to yield a white solid.
(p-Meth ylp h en yl)eth a n e 1-h yd r oxy-2-tosyla te: Rf 0.51;
1H NMR (CDCl3) δ 7.76 (d, 2, Ar, J ) 8.3 Hz), 7.38-7.12 (m, 6,
Ar, Artosyl), 4.93 (dd, 1, R-CH, J R,â1,â2 ) 3.3, 5.1 Hz), 4.15-4.00
(m, 2, â-CH1,2), 2.54 (br s, 1, OH), 2.44 (s, 3, CH3), 2.32 (s, 3,
CH3); +ve EI-MS m/z 306.093 71 ([M]+; calcd for C16H18O4S,
306.092 58).
(p-Br om op h en yl)eth a n e 1-h yd r oxy-2-tosyla te: Rf 0.64;
1H NMR (CDCl3) δ 7.75 (d, 2, Ar, J ) 8.3 Hz), 7.48-7.17 (m, 6,
Ar, Artosyl), 4.95 (dd, 1, R-CH, J R,â1,â2 ) 3.2, 4.4 Hz), 4.16-3.97
(m, 2, â-CH1,2), 2.52 (br s, 1, OH), 2.46 (s, 3, CH3); +ve EI-MS
m/z 371.981 39 ([M]+; calcd for C15H15O481BrS, 371.985 40),
369.988 40 ([M]+; calcd for C15H15O479BrS, 369.987 44).
(()-(p-Meth ylp h en yl)-1,2-eth a n ed iol: 4.46 g, 77%; Rf 0.17;
1H NMR (CDCl3) δ 7.22 (d, 2, Ar, J ) 8.1 Hz), 7.12 (d, 2, Ar, J
) 8.1 Hz), 4.71 (dd, 1, R-CH, J R,â1,â2 ) 3.5, 4.6 Hz), 3.68-3.55
(m, 2, â-CH1,2), 3.43 (br s, 2, OH), 2.32 (s, 3, CH3); +ve EI-MS
m/z 152.082 46 ([M]+; calcd for C9H12O2, 152.083 73).
Syn th esis of P a r a -Su bstitu ted Styr en e Oxid es. Para-
substituted phenylethane 1-hydroxy-2-tosylate (1.30 mmol, 1
equiv) and sodium hydroxide (105 mg, 2.60 mmol, 2 equiv) were
dissolved in methanol (20 mL) and stirred for 2 h at room
temperature. The reaction mixture was concentrated in vacuo
and water (20 mL) added. The product was extracted with
diethyl ether (3 × 30 mL), and the combined organic phases
were dried over magnesium sulfate and concentrated in vacuo.
The product was purified by wet flash column chromatography
(silica, eluted with a gradient of 0-5% ethyl acetate in hexane)
to yield the product as a pale yellow liquid (yield 72-89%).
p-Meth ylstyr en e oxid e: Rf 0.95; 1H NMR (CDCl3) δ 7.16
(s, 4, Ar), 3.82 (dd, 1, R-CH, J R,â2 ) 2.6 Hz, J R,â1 ) 4.1 Hz), 3.11
(dd, 1, â-CH1, J â1,R ) 4.1 Hz, J â1,â2 ) 5.5 Hz), 2.78 (dd, 1, â-CH2,
J â2,R ) 2.6 Hz, J â2,â1 ) 5.5 Hz), 2.34 (s, 3, CH3); +ve EI-MS m/z
134.073 32 ([M]+; calcd for C9H10O, 134.073 16).
(()-(p-Br om op h en yl)-1,2-eth a n ed iol: 6.47 g, 78%; Rf 0.21;
1H NMR (CDCl3) δ 7.47 (d, 2, Ar, J ) 8.5 Hz), 7.24 (d, 2, Ar, J
) 8.2 Hz), 4.78 (dd, 1, R-CH, J R,â1,â2 ) 3.5, 4.4 Hz), 3.78-3.55
(m, 2, â-CH1,2), 2.74 (br s, 1, OH), 2.21 (br s, 1, OH); +ve EI-MS
m/z 217.978 17 ([M]+; calcd for C8H9O281Br, 217.976 54),
215.980 35 ([M]+; calcd for C8H9O279Br, 215.978 59).
Syn th esis of (R)-P a r a -Su bstitu ted P h en yl-1,2-eth a n e-
d iols. These were prepared by the methods used for (()-para-
substituted phenyl-1,2-ethanediols but with the addition of
(DHQD)2PHAL (272 mg, 0.38 mmol, 0.01 equiv) along with the
potassium osmate dihydrate (ee >95%).
(R)-(p-Meth ylp h en yl)-1,2-eth a n ed iol: 3.87 g, 67%; Rf 0.17;
1H NMR (CDCl3) δ 7.18 (d, 2, Ar, J ) 8.3 Hz), 7.12 (d, 2, Ar, J
) 8.3 Hz), 4.70 (dd, 1, R-CH, J R,â1,â2 ) 3.5, 4.7 Hz), 3.67-3.54
(m, 2, â-CH1,2), 3.31 (br s, 2, OH), 2.32 (s, 3, CH3); +ve EI-MS
m/z 152.084 83 ([M]+; calcd for C9H12O2, 152.083 73).
p-Br om ostyr en e oxid e: Rf 0.98; 1H NMR (CDCl3) δ 7.47
(d, 2, Ar, J ) 8.5 Hz), 7.15 (d, 2, Ar, J ) 8.4 Hz), 3.82 (dd, 1,
R-CH, J R,â2 ) 2.6 Hz, J R,â1 ) 4.0 Hz), 3.14 (dd, 1, â-CH1, J â1,R
)
4.1 Hz, J â1,â2 ) 5.5 Hz), 2.75 (dd, 1, â-CH2, J â2,R ) 2.5 Hz, J â2,â1
) 5.5 Hz); +ve EI-MS m/z 199.965 67 ([M]+; calcd for C8H7O81
-
(R)-(p-Br om op h en yl)-1,2-eth a n ed iol: 6.05 g, 73%; Rf 0.21;
1H NMR (CDCl3) δ 7.49 (d, 2, Ar, J ) 8.4 Hz), 7.25 (d, 2, Ar, J
) 8.4 Hz), 4.80 (dd, 1, R-CH, J R,â1,â2 ) 3.6, 4.4 Hz), 3.80-3.57
(m, 2, â-CH1,2), 2.51 (br s, 2, OH); +ve EI-MS m/z 217.977 59
([M]+; calcd for C8H9O281Br, 217.976 54), 215.979 33 ([M]+; calcd
for C8H9O279Br, 215.978 59).
Br, 199.965 98), 197.967 48 ([M]+; calcd for C8H7O79Br,
197.968 03).
Syn th esis of P a r a -Su bstitu ted P h en yl-1,2-eth a n e Cyclic
Car bon ates. Para-substituted phenyl-1,2-ethanediol (4.6 mmol,
1 equiv) and sodium hydroxide (18 mg, 0.5 mmol, 0.1 equiv) were
suspended in dimethyl carbonate (10 mL, 10 g, 118 mmol, 25
equiv) and stirred under reflux for 24 h. The reaction mixture
was concentrated in vacuo, and the product was purified by wet
flash column chromatography (silica, eluted with a gradient of
0-30% ethyl acetate in hexane). The product was obtained as
a pale yellow solid (yield 71-98%).
Syn t h esis of (S)-P a r a -Su b st it u t ed P h en yl-1,2-et h a n e-
d iols. These were prepared by the methods used for (()-para-
substituted phenyl-1,2-ethanediols but with the addition of
(DHQ)2PHAL (272 mg, 0.38 mmol, 0.01 equiv) along with the
potassium osmate hydrate (ee >95%).
(S)-(p-Meth ylp h en yl)-1,2-eth a n ed iol: 4.27 g, 74%; Rf 0.17;
1H NMR (CDCl3) δ 7.20 (d, 2, Ar, J ) 8.1 Hz), 7.12 (d, 2, Ar, J
) 8.1 Hz), 4.72 (dd, 1, R-CH, J R,â1,â2 ) 3.6, 4.6 Hz), 3.69-3.55
(m, 2, â-CH1,2), 3.41 (br s, 1, OH), 3.11 (br s, 1, OH), 2.32 (s, 3,
(p-Meth ylp h en yl)-1,2-eth a n e cyclic ca r bon a te: Rf 0.40;
1H NMR (CDCl3) δ 7.26 (s, 4, Ar), 5.64 (t, 1, R-CH, J R,â1,â2 ) 8.0
Hz), 4.77 (dd, 1, â-CH1, J â1,R ) 8.2 Hz, J â1,â2 ) 8.5 Hz), 4.34 (dd,

46 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Barlow and Dipple
1, â-CH2, J â2,R ) 8.0 Hz, J â2,â1 ) 8.6 Hz), 2.38 (s, 3, CH3); +ve
EI-MS m/z 178.062 44 ([M]+; calcd for C10H10O3, 178.062 99).
(p-Br om op h en yl)-1,2-eth a n e cyclic ca r bon a te: Rf 0.55;
1H NMR (CDCl3) δ 7.59 (d, 2, Ar, J ) 8.5 Hz), 7.24 (d, 2, Ar, J
) 8.5 Hz), 5.64 (t, 1, R-CH, J R,â1,â2 ) 8.1 Hz), 4.80 (t, 1, â-CH1,
J â1,R ) 8.6 Hz), 4.30 (dd, 1, â-CH2, J â2,R ) 7.8 Hz, J â2,â1 ) 8.6
Hz); +ve EI-MS m/z 243.958 18 ([M]+; calcd for C9H7O381Br,
243.955 81), 241.959 56 ([M]+; calcd for C9H7O379Br, 241.957 86).
Syn th esis of P a r a -Su bstitu ted P h en yl-1-a zid oeth a n -2-
ols. Para-substituted phenyl-1,2-ethane cyclic carbonate (2.5
mmol, 1 equiv), sodium azide (322 mg, 5 mmol, 2 equiv), and
water (44 mL, 2.5 mmol, 1 equiv) were dissolved in DMF (30
mL) and stirred at 70 °C for 48 h. The reaction mixture was
concentrated in vacuo and water (50 mL) added. The product
was extracted into the organic phase with ethyl acetate (3 × 50
mL), dried over magnesium sulfate, filtered, and concentrated
in vacuo to yield a white solid (yield 81-86%).
1, R-CH, J R,â1 ) 3.8 Hz, J R,â2 ) 8.9 Hz), 4.78 (dd, 1, â-CH1, J â1,R
) 3.4 Hz, J â1,â2 ) 13.2 Hz), 4.56 (t, 1, H2′, J ) 4.8 Hz), 4.36 (dd,
1, â-CH2, J â2,R ) 8.9 Hz, J â2,â1 ) 13.2 Hz), 4.24 (t, 1, H3′, J ) 4.6
Hz), 4.17-4.16 (m, 1, H4′), 3.92 (dd, 1, H5′a, J ) 2.5, 12.3 Hz),
3.75 (dd, 1, H5′b, J ) 2.5, 12.3 Hz), 2.31 (s, 3, CH3); +ve FAB-
MS m/z 418.1770 ([M + H]+; calcd for C19H24N5O6, 418.1726).
SâN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 8.55 (s, 1, H-8), 7.34 (d, 2, Ar, J ) 7.9 Hz),
7.18 (d, 2, Ar, J ) 7.7 Hz), 5.89 (d, 1, H1′, J ) 4.6 Hz), 5.13 (dd,
1, R-CH, J R,â1 ) 3.5 Hz, J R,â2 ) 8.7 Hz), 4.78 (dd, 1, â-CH1, J â1,R
) 3.8 Hz, J â1,â2 ) 13.5 Hz), 4.56 (t, 1, H2′, J ) 4.7 Hz), 4.36 (dd,
1, â-CH2, J â2,R ) 8.9 Hz, J â2,â1 ) 13.3 Hz), 4.24 (t, 1, H3′, J ) 4.7
Hz), 4.18-4.16 (m, 1, H4′), 3.92 (dd, 1, H5′a, J ) 2.5, 12.5 Hz),
3.75 (dd, 1, H5′b, J ) 2.6, 12.4 Hz), 2.32 (s, 3, CH3); +ve FAB-
MS m/z 418.1777 ([M + H]+; calcd for C19H24N5O6, 418.1726).
RâN-7Br SOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 9.01 (s, 1, H-8), 7.48 (d, 2, Ar, J ) 8.3 Hz),
7.29 (d, 2, Ar, J ) 8.5 Hz), 6.43 (br s, 1, NH, exchanges with
D2O), 5.78 (d, 1, H1′, J ) 4.9 Hz), 5.19 (d, 1, R-CH, J ) 7.9 Hz),
4.70 (dd, 1, â-CH1, J â1,R ) 9.0 Hz, J â1,â2 ) 13.1 Hz), 4.45 (t, 1,
H2′, J ) 4.8 Hz), 4.16 (dd, 1, â-CH2, J â2,R ) 7.9 Hz, J â2,â1 ) 13.1
Hz), 4.11 (t, 1, H3′, J ) 4.3 Hz), 3.98 (dd, 1, H4′, J ) 3.6, 7.4 Hz),
3.71 (dd, 1, H5′a, J ) 2.7, 12.0 Hz), 3.58 (dd, 1, H5′b, J ) 2.7,
12.0 Hz); +ve FAB-MS m/z 482.0631 ([M + H]+; calcd for
(p -Met h ylp h en yl)-1-a zid oet h a n -2-ol: Rf 0.47; νmax/cm-1
1
2102 (N3); H NMR (CDCl3) δ 7.21 (s, 4, Ar), 4.63 (t, 1, R-CH,
J R,â1,â2 ) 6.5 Hz), 3.73 (d, 2, â-CH1,2, J â1,â2,R ) 6.5 Hz), 2.36 (s, 3,
CH3); +ve EI-MS m/z 177.089 62 ([M]+; calcd for C9H11N3O,
177.090 21).
(p -Br om op h en yl)-1-a zid oet h a n -2-ol: Rf 0.56; νmax/cm-1
1
2102 (N3); H NMR (CDCl3) δ 7.54 (d, 2, Ar, J ) 8.5 Hz), 7.23
C
18H21N5O679Br, 482.0674).
SâN-7Br SOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
(d, 2, Ar, J ) 8.5 Hz), 4.64 (dd, 1, R-CH, J R,â1 ) 5.4 Hz, J R,â2
)
7.2 Hz), 3.73 (t, 2, â-CH1,2, J â1,â2,R ) 5.8 Hz); +ve EI-MS m/z
240.986 28 ([M]+; calcd for C8H8N3O79Br, 240.985 07).
NMR (DMSO-d6) δ 9.01 (s, 1, H-8), 7.59 (d, 2, Ar, J ) 8.3 Hz),
7.43 (d, 2, Ar, J ) 8.3 Hz), 6.41 (br s, 1, NH, exchanges with
D2O), 5.82 (d, 1, H1′, J ) 4.8 Hz), 5.09 (d, 1, R-CH, J ) 8.7 Hz),
4.73 (dd, 1, â-CH1, J â1,R ) 2.3 Hz, J â1,â2 ) 13.2 Hz), 4.45 (t, 1,
H2′, J ) 4.8 Hz), 4.16 (dd, 1, â-CH2, J â2,R ) 9.2 Hz, J â2,â1 ) 13.2
Hz), 4.11 (t, 1, H3′, J ) 4.4 Hz), 3.99 (dd, 1, H4′, J ) 3.5, 7.4 Hz),
3.71 (dd, 1, H5′a, J ) 2.6, 12.0 Hz), 3.58 (dd, 1, H5′b, J ) 11.5
Hz); +ve FAB-MS m/z 482.0620 ([M + H]+; calcd for C18H21N5O6-
79Br, 482.0674).
Syn th esis of 7-[2-Hydr oxy-1-(p-m eth ylph en yl)eth yl]gu a-
n osin es (RrN-7MeSOG a n d SrN-7MeSOG) a n d 7-[2-Hy-
d r oxy-1-(p-br om op h en yl)eth yl]gu a n osin es (RrN-7Br SOG
a n d SrN-7Br SOG). Guanosine (450 mg, 1.60 mmol, 1 equiv)
and para-substituted styrene oxide (3.2 mmol, 2 equiv) were
dissolved in glacial acetic acid (50 mL), and the solution was
stirred, in the dark, at 37 °C for 16 h. The reaction mixture
was slowly added to diethyl ether (450 mL) with stirring, and
the resulting precipitate was collected by filtration. The solid
was dissolved in methanol (30 mL), applied to a Sephadex LH-
20 column (2.8 × 80 cm), and eluted at 1 mL/min with methanol.
Absorption of the eluate was monitored continuously at 254 nm,
and 8-mL fractions were collected. The product eluted in
fractions 34-45 and was identified by comparison of the UV
spectra under neutral, basic and acidic conditions with those of
literature spectra for 7-substituted guanosines (26), and those
fractions were pooled. The product was further purified by
reversed-phase HPLC eluting isocratically with 14% methanol
for RRN-7MeSOG (36 min), 14% methanol for SRN-7MeSOG
(57 min), 30% methanol for RRN-7BrSOG (21 min), or 35%
methanol for SRN-7BrSOG (23 min) in 50 mM ammonium
formate, pH 5.7.
Syn th esis of P a r a -Su bstitu ted P h en ylglycin ols. Para-
substituted phenyl-1-azidoethan-2-ol (2 mmol, 1 equiv) was
dissolved in anhydrous toluene (25 mL), and Red-Al (1 mL, 3.2
mmol, 1.6 equiv) was added dropwise with stirring, under
anhydrous conditions, at room temperature. The reaction
mixture was heated under reflux for 2 h and concentrated in
vacuo. Water (50 mL) was added slowly, and the product was
extracted into the organic phase with dichloromethane (3 × 50
mL), dried over magnesium sulfate, filtered, and concentrated
in vacuo to yield a white solid (yield 69-74%).
(p-Meth ylp h en yl)glycin ol: 1H NMR (CDCl3) δ 7.23-7.15
(m, 4, Ar), 4.00 (br s, 1, R-CH), 3.56-3.53 (m, 2, â-CH1,2), 2.34
(s, 3, CH3); +ve EI-MS m/z 151.099 70 ([M]+; calcd for C9H13
NO, 151.099 71).
-
(p-Br om op h en yl)glycin ol: 1H NMR (CDCl3) δ 7.45-7.30
(m, 4, Ar), 4.74 (t, 1, R-CH, J ) 5.4 Hz), 3.89-3.82 (m, 1, â-CH1),
3.49-3.39 (m, 1, â-CH2), 3.31 (br s, 3, NH2, OH); +ve EI-MS
m/z 214.995 09 ([M]+; calcd for C8H10NO79Br, 214.994 57),
216.992 98 ([M]+; calcd for C8H10NO81Br, 216.992 52).
Syn th esis of 7-[2-Hydr oxy-2-(p-m eth ylph en yl)eth yl]gu a-
n osin es (RâN-7MeSOG a n d SâN-7MeSOG) a n d 7-[2-Hy-
d r oxy-2-(p-br om op h en yl)eth yl]gu a n osin es (RâN-7Br SOG
a n d SâN-7Br SOG). Guanosine (450 mg, 1.60 mmol, 1 equiv)
and ammonium acetate (100 mg) were dissolved in 50% (v/v)
ethanol (100 mL), and optically active para-substituted styrene
oxide (3.2 mmol, 2 equiv) was added. The mixture was stirred
in the dark, at 37 °C for 96 h. The reaction mixture was
concentrated to dryness, and the solid residue was washed with
diethyl ether (50 mL) before resuspension in methanol (30 mL).
The suspension was filtered, and the filtrate was applied to a
Sephadex LH-20 column (2.8 × 80 cm) eluted at 1 mL/min with
methanol. Absorption of the eluate was monitored continuously
at 254 nm, and 8-mL fractions were collected. The product
eluted in fractions 45-56 and was identified by comparison of
the UV spectra under neutral, basic, and acidic conditions with
those of literature spectra for 7-substituted guanosines (26), and
those fractions were pooled. The product was further purified
by reversed-phase HPLC eluted isocratically with 14% methanol
for RâN-7MeSOG (46 min), 14% methanol for SâN-7MeSOG (32
min), 25% methanol for RâN-7BrSOG (30 min), or 22% methanol
for SâN-7BrSOG (48 min) in 50 mM ammonium formate, pH
5.7.
RrN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 9.38 (s, 1, H-8), 7.30 (d, 2, Ar, J ) 8.1 Hz),
7.18 (d, 2, Ar, J ) 7.9 Hz), 6.50 (dd, 1, R-CH, J ) 4.7 Hz), 5.82
(d, 1, H1′, J ) 4.7 Hz), 4.53 (t, 1, H2′, J ) 4.8 Hz), 4.12 (t, 1, H3′,
J ) 4.6 Hz), 4.01-3.98 (m, 1, H4′), 3.76-3.51 (m, 4, â-CH1,2
5′a,b), 2.31 (s, 3, CH3); +ve FAB-MS m/z 440.1529 ([M + Na]+;
calcd for C19H23N5O6Na, 440.1545).
,
H
SrN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 9.41 (s, 1, H-8), 7.36 (d, 2, Ar, J ) 7.9 Hz),
7.21 (d, 2, Ar, J ) 8.0 Hz), 6.47 (dd, 1, R-CH, J ) 4.4 Hz), 5.93
(d, 1, H1′, J ) 4.1 Hz), 4.60 (t, 1, H2′, J ) 4.6 Hz), 4.33-4.27 (m,
2, H3′, â-CH1), 4.18-4.14 (m, 2, H4′, â-CH2), 3.95 (dd, 1, H5′a, J
) 2.2, 12.4 Hz), 3.78 (dd, 1, H5′b, J ) 1.8, 12.4 Hz), 2.31 (s, 3,
CH3); +ve FAB-MS m/z 440.1586 ([M + Na]+; calcd for
C19H23N5O6Na, 440.1546).
RâN-7MeSOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 8.55 (s, 1, H-8), 7.34 (d, 2, Ar, J ) 8.0 Hz),
7.18 (d, 2, Ar, J ) 7.8 Hz), 5.89 (d, 1, H1′, J ) 4.6 Hz), 5.13 (dd,

Mechanisms of Guanosine Aralkylation
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 47
RrN-7Br SOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 9.35 (s, 1, H-8), 7.59 (d, 2, Ar, J ) 8.5 Hz),
7.39 (d, 2, Ar, J ) 8.5 Hz), 6.45 (dd, 1, R-CH, J R,â2 ) 4.8 Hz,
J R,â1 ) 8.0 Hz), 5.82 (d, 1, H1′, J ) 4.6 Hz), 4.54 (t, 1, H2′, J )
4.6 Hz), 4.27 (dd, 1, â-CH1, J â1,R ) 8.1 Hz, J â1,â2 ) 11.8 Hz),
4.18 (t, 1, H3′, J ) 4.5 Hz), 4.04 (dd, 1, â-CH2, J R2,R ) 4.8 Hz,
J â2,â1 ) 12.0 Hz), 4.00 (dd, 1, H4′, J ) 3.0, 7.2 Hz) 3.73 (dd, 1,
3.54-3.50 (m, 1, H5′b); +ve FAB-MS m/z 504.0460 ([M + Na]+;
calcd for C18H19N5O679BrNa, 504.0415).
Samples of the N2-[2-hydroxy-1-(p-substituted phenyl)eth-
yl]guanosines were converted to N2-[2-hydroxy-1-(p-substituted
phenyl)ethyl]guanines by acid hydrolysis with 1 M HCl at 100
°C for 1 h. The products were purified by reversed-phase HPLC
eluting with 35% methanol for N2-[2-hydroxy-1-(p-methylphen-
yl)ethyl]guanines (34 min) or 45% methanol for N2-[2-hydroxy-
1-(p-bromophenyl)ethyl]guanines (22 min) in water.
H
5′a, J ) 2.9, 12.2 Hz), 3.78 (d, 1, H5′b, J ) 12.2 Hz); +ve FAB-
MS m/z 482.0674 ([M + H]+; calcd for C18H20N5O679Br, 482.0596).
SrN-7Br SOG: UV λmax (methanol) 259, 282 (sh) nm; 1H
NMR (DMSO-d6) δ 9.34 (s, 1, H-8), 7.58 (d, 2, Ar, J ) 8.5 Hz),
7.39 (d, 2, Ar, J ) 8.5 Hz), 6.49-6.46 (m, 1, R-CH), 5.82 (d, 1,
H1′, J ) 4.8 Hz), 4.34 (t, 1, H2′, J ) 4.9 Hz), 4.30-4.22 (m, 1,
â-CH1), 4.09 (dd, 1, H3′, J ) 5.0, 10.1 Hz), 4.05-4.00 (m, 2,
â-CH2, H4′), 3.61-3.55 (m, 1, H5′a), 3.47-3.41 (m, 1, H5′b); +ve
FAB-MS m/z 482.0664 ([M + H]+; calcd for C18H20N5O679Br,
482.0596).
Syn th esis of In d ivid u a l Dia ster eom er s of N2-[2-Hy-
d r oxy-1-(p-m eth ylp h en yl)eth yl]gu a n in es (RrN2MeSOGu a
a n d SrN2MeSOGu a ) a n d N2-[2-Hyd r oxy-1-(p-br om op h en -
yl)eth yl]gu a n in es (RrN2Br SOGu a a n d SrN2Br SOGu a ).
2-Chloro-6-hydroxypurine (100 mg, 0.6 mmol, 1 equiv) and
optically active para-substituted phenylglycinol (1.2 mmol, 2
equiv) were dissolved in DMSO (4 mL) and stirred at 90 °C for
164 h. The reaction mixture was concentrated in vacuo,
suspended in methanol (10 mL), and filtered. The product was
purified from the filtrate by reversed-phase HPLC eluting with
35% methanol for RRN2MeSOGua (34 min) and SRN2MeSOGua
(34 min) or 45% methanol for RRN2BrSOGua (22 min) and SRN2-
BrSOGua (22 min) in water.
RrN2MeSOGu a : UV λmax (methanol) 249, 279 nm; 1H NMR
(DMSO-d6) δ 10.51 (br s, 1, NH, exchanges with D2O), 7.60 (s,
1, H-8), 7.21 (d, 2, Ar, J ) 7.9 Hz), 7.12 (d, 2, Ar, J ) 8.0 Hz),
4.90 (m, 1, R-CH), 3.72-3.69 (m, 1, â-CH1), 3.60-3.57 (m, 1,
â-CH2), 2.26 (s, 3, CH3); +ve FAB-MS m/z 286.1322 ([M + H]+;
calcd for C14H16N5O2, 286.1304).
SrN2MeSOGu a : UV λmax (methanol) 249, 279 nm; 1H NMR
(DMSO-d6) δ 7.64 (s, 1, H-8), 7.21 (d, 2, Ar, J ) 8.0 Hz), 7.12 (d,
2, Ar, J ) 7.9 Hz), 4.91-4.90 (m, 1, R-CH), 3.72-3.69 (m, 1,
â-CH1), 3.61-3.57 (m, 1, â-CH2), 2.26 (s, 3, CH3); +ve EI-MS
m/z 285.121 92 ([M]+; calcd for C14H15N5O2, 285.122 58).
RrN2Br SOGu a : UV λmax (methanol) 251, 276 nm; 1H NMR
(DMSO-d6) δ 7.61 (s, 1, H-8), 7.50 (d, 2, Ar, J ) 8.4 Hz), 7.29 (d,
2, Ar, J ) 8.4 Hz), 4.90-4.89 (m, 1, R-CH), 3.73-3.70 (m, 1,
â-CH1), 3.62-3.59 (m, 1, â-CH2); +ve FAB-MS m/z 350.0146 ([M
+ H]+; calcd for C13H13N5O279Br, 350.0126).
Syn th esis of N2-[2-Hydr oxy-1-(p-m eth ylph en yl)eth yl]gu a-
n osin es (RrN2MeSOG a n d SrN2MeSOG) a n d N2-[2-Hy-
d r oxy-1-(p-br om op h en yl)eth yl]gu a n osin es (R rN2Br SOG
a n d SrN2Br SOG). Guanosine (150 mg, 0.5 mmol, 1 equiv) and
sodium carbonate (200 mg, 2 mmol, 4 equiv) were dissolved in
water (10 mL), and the solution was stirred at 90 °C for 3 h.
Para-substituted styrene oxide (1 mmol, 2 equiv) was added,
and the mixture was stirred at 90 °C for an additional 72 h.
The reaction mixture was allowed to cool before addition of
methanol (12 mL). The resulting solution was applied to a
Sephadex LH-20 column (2.8 × 80 cm) and eluted at 1 mL/min
with 40% (v/v) methanol. Absorption of the eluate was moni-
tored continuously at 254 nm, and 8-mL fractions were collected.
The product eluted in fractions 87-103 and was identified by
comparison of the UV spectra under neutral, basic, and acidic
conditions with those of literature spectra for N2-substituted
guanosines (18), and those fractions were pooled. The product
was further purified by reversed-phase HPLC eluting isocrati-
cally with 25% methanol for RRN2MeSOG (22 min) and SRN2-
MeSOG (28 min) and 30% methanol for RRN2BrSOG (25 min)
and SRN2BrSOG (47 min) in water.
RrN2MeSOG: UV λmax (methanol) 256, 276 (sh) nm; 1H
NMR (DMSO-d6) δ 10.79 (br s, 1, NH, exchanges with D2O),
7.88 (s, 1, H-8), 7.34 (br s, 1, NH, exchanges with D2O), 7.20 (d,
2, Ar, J ) 7.9 Hz), 7.10 (d, 2, Ar, J ) 8.1 Hz), 5.63 (d, 1, H1′, J
) 5.5 Hz), 4.99-4.97 (m, 1, R-CH), 4.49-4.46 (m, 1, H2′), 4.39-
4.37 (m, 1, H3′), 4.13-4.10 (m, 1, H4′), 3.74-3.70 (m, 1, â-CH1),
3.66-3.63 (m, 2, â-CH2, H5′a), 3.57-3.53 (m, 1, H5′b), 2.29 (s, 3,
CH3); +ve FAB-MS m/z 440.1592 ([M + Na]+; calcd for
C19H23N5O6Na, 440.1546).
SrN2MeSOG: UV λmax (methanol) 256, 276 (sh) nm; 1H NMR
(DMSO-d6) δ 10.85 (br s, 1, NH, exchanges with D2O), 7.88 (s,
1, H-8), 7.31 (br s, 1, NH, exchanges with D2O), 7.27 (d, 2, Ar,
J ) 7.9 Hz), 7.15 (d, 2, Ar, J ) 8.1 Hz), 5.63 (d, 1, H1′, J ) 5.8
Hz), 4.96 (dd, 1, R-CH, J ) 5.4, 12.4 Hz), 4.39-4.38 (m, 1, H2′),
4.13-4.11 (m, 1, H3′), 4.13-4.10 (m, 1, H4′), 3.72 (dd, 1, â-CH1,
J ) 4.4, 10.6 Hz), 3.66-3.63 (m, 2, H5′a, â-CH2), 3.57-3.53 (m,
1, H5′b), 2.29 (s, 3, CH3); +ve FAB-MS m/z 462.1369 ([M + Na2]+;
calcd for C19H23N5O6Na2, 462.1366).
RrN2Br SOG: UV λmax (methanol) 247, 278 (sh) nm; 1H NMR
(DMSO-d6) δ 10.55 (br s, 1, NH, exchanges with D2O), 7.90 (s,
1, H-8), 7.52 (d, 2, Ar, J ) 8.4 Hz), 7.34 (d, 2, Ar, J ) 8.4 Hz),
7.11 (br s, 1, NH, exchanges with D2O), 5.63 (d, 1, H1′, J ) 5.4
Hz), 5.00 (dd, 1, R-CH, J ) 5.0, 9.1 Hz), 4.42 (dd, 1, H2′, J ) 5.8,
11.0 Hz), 4.04 (dd, 1, H3′, J ) 4.1, 8.4 Hz), 3.82 (dd, 1, H4′, J )
4.3, 8.7 Hz), 3.76-3.73 (m, 1, â-CH1), 3.68-3.64 (m, 1, â-CH2),
3.53-3.49 (m, 1, H5′a), 3.34-3.29 (m, 1, H5′b); +ve FAB-MS m/z
504.0450 ([M + Na]+; calcd for C18H19N5O679BrNa, 504.0415).
SrN2Br SOG: UV λmax (methanol) 247, 278 (sh) nm; 1H NMR
(DMSO-d6) δ 10.62 (br s, 1, NH, exchanges with D2O), 7.87 (s,
1, H-8), 7.51 (d, 2, Ar, J ) 8.4 Hz), 7.32 (d, 2, Ar, J ) 8.3 Hz),
7.23 (br s, 1, NH, exchanges with D2O), 5.58 (d, 1, H1′, J ) 5.8
Hz), 4.95-4.93 (m, 1, R-CH), 4.33 (dd, 1, H2′, J ) 5.5, 10.5 Hz),
4.07 (dd, 1, H3′, J ) 4.1, 10.0 Hz), 3.84 (dd, 1, H4′, J ) 4.1, 8.0
Hz), 3.75-3.71 (m, 1, â-CH1), 3.64-3.59 (m, 2, â-CH2, H5′a),
SrN2Br SOGu a : UV λmax (methanol) 251, 276 nm; 1H NMR
(DMSO-d6) δ 7.64 (s, 1, H-8), 7.51 (d, 2, Ar, J ) 8.4 Hz), 7.30 (d,
2, Ar, J ) 8.4 Hz), 4.92-4.91 (m, 1, R-CH), 3.72-3.71 (m, 1,
â-CH1), 3.62-3.60 (m, 1, â-CH2); +ve FAB-MS m/z 350.0209 ([M
+ H]+; calcd for C12H13N5O279Br, 350.0126).
The N2-[2-hydroxy-1-(p-methylphenyl)ethyl]guanines (RRN2-
MeSOGua and SRN2MeSOGua) and N2-[2-hydroxy-1-(p-bro-
mophenyl)ethyl]guanines (RRN2BrSOGua and SRN2BrSOGua)
were used to assign the regio- and stereochemistry of the N2-
[2-hydroxy-1-(p-methylphenyl)ethyl]guanosines (RRN2MeSOG
and SRN2MeSOG) and N2-[2-hydroxy-1-(p-bromophenyl)eth-
yl]guanosines (RRN2BrSOG and SRN2BrSOG) after the gua-
nosines had been converted to guanines by acid hydrolysis by
comparison of the 1H NMR and CD spectra.
Syn th esis of O6-[2-Hydr oxy-1-(p-m eth ylph en yl)eth yl]gu a-
n osin es (RrO6MeSOG a n d SrO6MeSOG), O6-[2-Hyd r oxy-
2-(p-m eth ylp h en yl)eth yl]gu a n osin es (RâO6MeSOG a n d
SâO6MeSOG), O6-[2-Hyd r oxy-1-(p-br om op h en yl)eth yl]gu a -
n osin es (RrO6Br SOG a n d SrO6Br SOG), a n d O6-[2-Hy-
d r oxy-2-(p-br om op h en yl)et h yl]gu a n osin es (RâO6Br SOG
a n d SâO6Br SOG). Optically active para-substituted phenyle-
thanediol (4 mmol, 10 equiv) was melted at >100 °C, and sodium
(23 mg, 1 mmol, 2.5 equiv) was added. After the sodium had
dissolved, 2-amino-6-chloropurine riboside (120 mg, 0.4 mmol,
1 equiv) was added, and the reaction mixture was stirred at
>100 °C for 96 h. The reaction mixture was allowed to cool
and methanol (10 mL) added. The suspension was filtered, the
filtrate was added to water (10 mL), and the resulting solution
was applied to a Sephadex LH-20 column (2.8 × 80 cm) which
was eluted at 1 mL/min with 50% (v/v) methanol. Absorption
of the eluate was monitored continuously at 254 nm, and 8-mL
fractions were collected. The products eluted in fractions 130-
155 and were identified by comparison of the UV spectra under
neutral conditions with those of literature spectra for O6-

48 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Barlow and Dipple
substituted guanosines (27), and those fractions were pooled.
The product was further purified by reversed-phase HPLC
eluting isocratically with 35% methanol for RRO6MeSOG (51
min) and RâO6MeSOG (54 min), 40% methanol for SRO6MeSOG
(26 min) and SâO6MeSOG (43 min), 35% methanol for RRO6-
BrSOG (79 min) and RâO6BrSOG (85 min), and 40% methanol
for SRO6BrSOG (30 min) and SâO6BrSOG (46 min) in water.
RrO6MeSOG: UV λmax (methanol) 248, 282 nm; 1H NMR
(DMSO-d6) δ 8.10 (s, 1, H-8), 7.35 (d, 2, Ar, J ) 8.0 Hz), 7.16 (d,
2, Ar, J ) 7.9 Hz), 6.34-6.31 (m, 3, NH2, exchanges with D2O
and R-CH), 5.76 (d, 1, H1′, J ) 6.0 Hz), 4.50-4.46 (m, 1, H2′),
4.09 (dd, 1, H3′, J ) 5.2, 10.4 Hz), 3.90-3.83 (m, 2, H4′, â-CH1),
3.73-3.68 (m, 1, â-CH2), 3.64-3.61 (m, 1, H5′a), 3.55-3.52 (m,
1, H5′b), 2.30 (s, 3, CH3); +ve FAB-MS m/z 440.1500 ([M + Na]+;
calcd for C19H23N5O6Na, 440.1546).
SrO6MeSOG: UV λmax (methanol) 248, 282 nm; 1H NMR
(DMSO-d6) δ 8.10 (s, 1, H-8), 7.32 (d, 2, Ar, J ) 8.0 Hz), 7.13 (d,
2, Ar, J ) 7.9 Hz), 6.33-6.31 (m, 3, NH2, exchanges with D2O
and R-CH), 5.76 (d, 1, H1′, J ) 6.0 Hz), 4.50-4.45 (m, 1, H2′),
4.11-4.08 (m, 1, H3′), 3.89-3.82 (m, 2, H4′, â-CH1), 3.73-3.68
(m, 1, â-CH2), 3.64-3.61 (m, 1, H5′a), 3.55-3.52 (m, 1, H5′b), 2.27
(s, 3, CH3); +ve FAB-MS m/z 440.1529 ([M + Na]+; calcd for
C19H23N5O6Na, 440.1546).
RâO6MeSOG: UV λmax (methanol) 248, 283 nm; 1H NMR
(DMSO-d6) δ 8.10 (s, 1, H-8), 7.35 (d, 2, Ar, J ) 7.9 Hz), 7.16 (d,
2, Ar, J ) 8.0 Hz), 6.42 (br s, 2, NH2, exchanges with D2O),
5.78 (d, 1, H1′, J ) 6.0 Hz), 4.97 (dd, 1, R-CH, J ) 4.0, 7.8 Hz),
4.45-4.35 (m, 3, H2′, â-CH1,2), 4.09 (dd, 1, H3′, J ) 5.1, 10.3 Hz),
3.89 (dd, 1, H4′, J ) 3.9, 7.5 Hz), 3.64-3.61 (m, 1, H5′a), 3.55-
3.51 (m, 1, H5′b), 2.30 (s, 3, CH3); +ve FAB-MS m/z 440.1523
([M + Na]+; calcd for C19H23N5O6Na, 440.1546).
SâO6MeSOG: UV λmax (methanol) 248, 283 nm; 1H NMR
(DMSO-d6) δ 8.10 (s, 1, H-8), 7.35 (d, 2, Ar, J ) 8.1 Hz), 7.16 (d,
2, Ar, J ) 7.8 Hz), 6.42 (br s, 2, NH2, exchanges with D2O),
5.78 (d, 1, H1′, J ) 6.0 Hz), 4.97 (dd, 1, R-CH, J ) 3.9, 7.8 Hz),
4.44-4.35 (m, 3, H2′, â-CH1,2), 4.09 (dd, 1, H3′, J ) 5.1, 10.3 Hz),
3.89 (dd, 1, H4′, J ) 3.8, 7.4 Hz), 3.64-3.61 (m, 1, H5′a), 3.55-
3.52 (m, 1, H5′b), 2.29 (s, 3, CH3); +ve FAB-MS m/z 440.1519
([M + Na]+; calcd for C19H23N5O6Na, 440.1546).
RrO6Br SOG: UV λmax (methanol) 248, 284 nm; 1H NMR
(DMSO-d6) δ 8.10 (s, 1, H-8), 7.55 (d, 2, Ar, J ) 8.3 Hz), 7.44 (d,
2, Ar, J ) 8.4 Hz), 6.44 (s, 2, NH2, exchanges with D2O), 6.29
(dd, 1, R-CH, J ) 4.4, 7.1 Hz), 5.78 (d, 1, H1′, J ) 6.0 Hz), 4.50
(t, 1, H2′), 4.10-4.09 (m, 1, H3′), 3.90-3.87 (m, 1, H4′), 3.86-
3.84 (m, 1, â-CH1), 3.76-3.72 (m, 1, â-CH2), 3.64-3.61 (m, 1,
Ar a lk yla tion of [14C]Gu a n osin e in Aqu eou s Solu tion by
Op tica lly Active P a r a -Su bstitu ted Styr en e Oxid es. Opti-
cally active para-substituted styrene oxide (5 mL) was added
to a solution of [14C]guanosine (1 mCi) in 50 mM Tris‚HCl buffer,
pH 7.0 (1 mL). The reaction mixture was stirred in the dark at
37 °C for 24 h. Aliquots (250 mL) were used to dissolve a
mixture containing guanosine, the four 7-substituted isomers,
the two N2-substituted isomers, and the four O6-substituted
isomers of the respective para-substituted styrene oxide adducts.
The mixture was separated by reversed-phase HPLC eluting
at 1 mL/min using the following solvents: A, 50 mM ammonium
formate, pH 5.7; B, methanol. The p-methylstyrene oxide-
guanosine adducts were resolved using the following gradient
of B in A: 0 min, 10%; 10 min, 10%; 60 min, 20%; 90 min, 20%;
130 min, 24%; 220 min, 33%. Typical retention times for the
individual products were as follows: guanosine, 9 min; SâN-
7MeSOG, 46 min; RRN-7MeSOG, 57 min; RâN-7MeSOG, 74
min; RRN2MeSOG, 78 min; SRN-7MeSOG, 108 min; SRN2-
MeSOG, 117 min; SRO6MeSOG, 155 min; SâO6MeSOG, 180
min; RRO6MeSOG, 183 min; RâO6MeSOG, 186 min. The
p-bromostyrene oxide-guanosine adducts were resolved using
the following gradient of B in A: 0 min, 20%; 80 min, 24%; 100
min, 32%; 240 min, 44%. Typical retention times for the
individual products were as follows: guanosine, 5 min; SâN-
7BrSOG, 92 min; RRN-7BrSOG, 108 min; RâN-7BrSOG, 122
min; RRN2BrSOG, 126 min; SRN-7BrSOG, 144 min; SRN2-
BrSOG, 153 min; SRO6BrSOG, 188 min; SâO6BrSOG, 212 min;
RRO6BrSOG, 215 min; RâO6BrSOG, 218 min. Fractions (1 mL)
were collected throughout the elution, and the radioactivity in
each fraction was determined by liquid scintillation counting.
Resu lts
Investigation of the effects of para-substituents on the
yields and configurations of various products formed in
reactions between guanosine and p-substituted styrene
oxides required the preparation of the anticipated prod-
ucts as markers, as well as the establishment of their
stereochemical configurations. Previous studies (18) with
unsubstituted styrene oxide indicated that markers for
five diastereomeric pairs of guanosine products would be
necessary (Chart 1). Oxirane ring opening at the R- or
â-carbons of each styrene oxide enantiomer by the ring
nitrogen at the guanosine 7-position can give rise to two
diastereomeric pairs, i.e., R- or SâN-7XSOG and R- or
SRN-7XSOG (Chart 1). Epoxide ring opening at the
R-carbon by the exocyclic N2- and O6-positions of gua-
nosine gives rise to two more pairs (R- and SRN2XSOG
and R- and SRO6XSOG, respectively), and equilibration
of the RO6-substituted products with âO6-substituted
products (27) requires preparation of R- and SâO6XSOG.
Thus the required p-bromo- and p-methyl-substituted
markers were prepared using strategies that allowed the
stereochemical outcome of the reactions to be determined.
Racemic p-bromo- and p-methylstyrene oxides were
prepared by established procedures (28). The synthesis
of optically active p-substituted epoxides employed asym-
metric dihydroxylation of p-substituted styrenes (29) to
produce optically active diols with an enantiomeric excess
of >95%. The epoxides were prepared from these diols
by tosylation of the primary hydroxyl group followed by
base-assisted ring closure with elimination of toluene-
sulfonic acid (28).
H
5′a), 3.55-3.50 (m, 1, H5′b); +ve FAB-MS m/z 482.0610 ([M +
H]+; calcd for C18H21N5O679Br, 482.0596).
SrO6Br SOG: UV λmax (methanol) 248, 284 nm; 1H NMR
(DMSO-d6) δ 8.12 (s, 1, H-8), 7.54 (d, 2, Ar, J ) 8.5 Hz), 7.39 (d,
2, Ar, J ) 8.5 Hz), 6.36 (s, 2, NH2, exchanges with D2O), 6.29
(dd, 1, R-CH, J ) 4.6, 7.2 Hz), 5.76 (d, 1, H1′, J ) 6.0 Hz), 4.48-
4.45 (m, 1, H2′), 4.10-4.08 (m, 1, H3′), 3.89-3.83 (m, 2, H4′,
â-CH1), 3.76-3.72 (m, 1, â-CH2), 3.64-3.60 (m, 1, H5′a), 3.54-
3.50 (m, 1, H5′b); +ve FAB-MS m/z 482.0635 ([M + H]+; calcd
for C18H21N5O679Br, 482.0596).
RâO6Br SOG: UV λmax (methanol) 248, 284 nm; 1H NMR
(DMSO-d6) δ 8.12 (s, 1, H-8), 7.53 (d, 2, Ar, J ) 8.3 Hz), 7.39 (d,
2, Ar, J ) 8.4 Hz), 6.38 (s, 2, NH2, exchanges with D2O), 5.77
(d, 1, H1′, J ) 6.0 Hz), 5.02-5.01 (m, 1, R-CH), 4.46-4.43 (m, 3,
H2′, â-CH1,2), 4.09 (dd, 1, H3′, J ) 5.0, 10.0 Hz), 3.88 (dd, H4′, J
) 3.8, 7.5 Hz), 3.55-3.51 (m, 1, H5′a), 3.50-3.46 (m, 1, H5′b);
+ve FAB-MS m/z 482.0633 ([M + H]+; calcd for C18H21N5O6-
79Br, 482.0596).
SâO6Br SOG: UV λmax (methanol) 248, 284 nm; 1H NMR
(DMSO-d6) δ 8.10 (s, 1, H-8), 7.55 (d, 2, Ar, J ) 8.4 Hz), 7.44 (d,
2, Ar, J ) 8.4 Hz), 6.43 (s, 2, NH2, exchanges with D2O), 5.78
(d, 1, H1′, J ) 6.0 Hz), 5.01 (dd, 1, R-CH, J ) 3.5, 7.1 Hz), 4.47-
4.43 (m, 3, H2′, â-CH1,2), 4.09 (dd, 1, H3′, J ) 5.2, 10.4 Hz), 3.90-
3.88 (dd, H4′), 3.65-3.61 (m, 1, H5′a), 3.56-3.51 (m, 1, H5′b); +ve
FAB-MS m/z 482.0630 ([M + H]+; calcd for C18H21N5O679Br,
482.0596).
The 7-substituted guanosine markers were prepared
from guanosine and optically active epoxides (Scheme 1).
Reactions in glacial acetic acid or 50% ethanol were
known to favor attack by the guanosine ring nitrogen at
either the R- (benzylic) or â-position of the epoxide,
respectively (18). Reactions in glacial acetic acid pro-

Mechanisms of Guanosine Aralkylation
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 49
Ch a r t 1. Dia ster eom er ic P a ir s of P r od u cts
An ticip a ted fr om Rea ction of p-Meth yl- a n d
p-Br om ostyr en e Oxid es w ith Gu a n osin e u n d er
Neu tr a l Aqu eou s Con d ition s
Sch em e 2. Syn th esis of O6-Su bstitu ted Styr en e
Oxid e-Gu a n osin e Ad d u cts
1
R- and â-carbons in H NMR spectra. The positive charge
on the ring nitrogen resulted in a downfield shift of the
adjacent alkyl protons, such that the proton on the
R-carbon shifted from around 5.1 ppm in the â-substi-
tuted compounds to around 6.5 ppm in the R-substituted
compounds. This change was also apparent, but less
dramatic, for the protons on the â-carbon that typically
shifted from between 4 and 3.5 ppm in the R-substituted
compounds to between 4.8 and 4.2 ppm in the â-substi-
tuted compounds.
Sch em e 1. Syn th esis of 7-Su bstitu ted Styr en e
Oxid e-Gu a n osin e Ad d u cts
The finding that optically active R-substituted products
were formed in each case is consistent with a bimolecular
reaction mechanism which gives rise to a product with
inversion of stereochemistry at the R-carbon. The alter-
native explanation that substitution at the chiral center
occurred with complete retention of configuration seems
unlikely, and the R-substituted products were therefore
assigned the inverse configuration of the starting epoxide.
Since formation of the â-substituted products does not
involve any bond-breaking or bond-making steps at the
chiral carbon, the products at this position must retain
the stereochemistry of the starting epoxide.
Synthesis of the O6-substituted markers was achieved
by displacement of chloride from 2-amino-6-chloropurine
riboside with alkoxides prepared from addition of sodium
to the optically active p-substituted diols. As formation
of the alkoxides and the subsequent reaction with the
chloropurine riboside do not involve any changes in
bonding at the chiral carbon of the diols, the configuration
of the starting diol is conferred upon the products. This
method produced markers of the RO6- and âO6-substi-
tuted products that would have arisen from aralkylation
of guanosine by p-substituted styrene oxides (Scheme 2).
It has been reported that these O6-substituted gua-
nosine-styrene oxide derivatives isomerize in a base-
catalyzed rearrangement (27), and as anticipated, the
analogous reaction products in this study also behaved
similarly. When a single isomer was treated with base,
an equilibrium mixture of two isomers was produced. The
duced optically active R-substituted products from both
the p-methyl- and p-bromostyrene oxides. In 50% etha-
nol, however, mixtures of R- and â-products were found
although each product constituted a single diastereomer.
â-Substituted products predominated, especially when
p-bromostyrene oxide was the substrate.
The R- and â-substituted regioisomers were readily
distinguished by the chemical shifts of the protons at the

50 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Barlow and Dipple
Sch em e 3. Syn th esis of N2-Su bstitu ted Styr en e Oxid e-Gu a n osin e a n d N2-Su bstitu ted Styr en e
Oxid e-Gu a n in e Ad d u cts
equilibrium ratio of R:â O6-substituted products was
found to be ∼1:1 for the p-methyl-substituted compounds
and ∼1:2 for the p-bromo-substituted compounds. After
separation by HPLC, the R- and â-substituted forms could
easily be distinguished by examination of the chemical
Thus, the R-diastereomer was identified as the product
which eluted earlier than the S-diastereomer in the
HPLC system used here.
Once all the markers were prepared and characterized,
HPLC conditions were developed to separate the markers
and their order of elution was established (Figure 2).
Separation of radioactive products [illustrated for (S)-p-
methylstyrene oxide-guanosine products in Figure 2C]
allowed the product stereochemistry and yields to be
evaluated in reactions of optically active p-substituted
styrene oxides and [14C]guanosine in aqueous solution.
The results from triplicate analyses with both the p-
methyl- and the p-bromostyrene oxides are summarized
in Table 1 [previously published data for reactions with
styrene oxide are included to aid comparison (18)].
The overall yield of products from both epoxides was
low. The p-bromostyrene oxides converted approximately
4% of the guanosine to products, whereas the p-methyl-
styrene oxides reacted more extensively, converting ap-
proximately 8% of the guanosine to products. Examina-
tion of the products formed in each reaction indicated
that the para-substituent had a marked effect not only
on the product yields but also on the distribution of
products at the 7-, N2-, and O6-positions of guanosine.
N2-Substituted adducts accounted for around 60% of the
total products formed in reactions involving the p-
methylstyrene oxides with the remainder mostly being
7-substituted compounds. This selectivity was reversed
in reactions involving the p-bromostyrene oxides where
nearly 85% of the total products formed were 7-substi-
tuted compounds and the remainder almost all N2-
substituted adducts. Neither the p-methyl- nor the
p-bromo-substituted epoxides formed substantial quanti-
ties of O6-substituted products. The chirality of the
epoxide did not display any appreciable effect on either
the product yields or the distributions.
1
shifts of the protons at the R- and â-carbons in the H
NMR spectra. These protons shifted in a manner similar
to that seen for the 7-substituted guanosines. The
electron-withdrawing effect of the oxygen conjugated with
the purine ring shifted the proton at the R-position from
around 5.0 ppm in the â-substituted compounds to
around 6.3 ppm in the R-substituted compounds. Simi-
larly, the protons on the â-carbon were shifted from
around 3.8 ppm in the R-substituted compounds to
around 4.4 ppm in the â-substituted compounds.
N2-Substituted markers were prepared by reactions of
racemic epoxides with guanosine under alkaline condi-
tions that are known to increase the reactivity of the
exocyclic nitrogen (16) (Scheme 3). The reactions with
the racemic epoxides gave only two N2-substituted gua-
nosines which, after purification and separation by
1
HPLC, were identified as diastereomers by their H NMR
spectra. The mechanism by which ionized guanosine
undergoes alkylation at the N2-position is unclear, and
therefore the regio- and stereochemistry of these products
was not immediately obvious.
Independent structure-defining syntheses of the cor-
responding RN2-substituted guanine enantiomers were
achieved, however, by the displacement of chloride from
2-chloro-6-hydroxypurine by each optically active p-
substituted phenylglycinol (Scheme 3). The necessary
glycinols were synthesized via a route developed by
Chang and Sharpless (30) in which optically active diols
were converted to cyclic carbonates and subsequently
ring-opened at the R-carbon, with inversion of stereo-
chemistry, by azide. Reduction then gave a glycinol of
1
known configuration. The CD spectra (Figure 1) and H
The stereochemical differences in products at the 7-,
N2-, and O6-positions indicated that these sites reacted
with the epoxide in different ways. Reactions at the
7-position for p-substituted styrene oxides gave products
with predominantly inverted stereochemistry. In con-
trast, reactions at the N2- and O6-positions gave products
of both inverted and retained stereochemistry, with the
NMR spectra of the guanine reference compounds pre-
pared in this fashion were compared to those of N2-
substituted guanines prepared from the nucleosides by
acid hydrolysis. This comparison allowed the marker
nucleoside products to be identified as R-substituted
compounds and their configurations to be assigned.

Mechanisms of Guanosine Aralkylation
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 51
F igu r e 2. HPLC separation of para-substituted styrene oxide-
guanosine adducts: panel A, separation of p-bromostyrene
oxide-guanosine adducts; panel B, separation of p-methylsty-
rene oxide-guanosine adducts. Peaks: 1, guanosine; 2, SâN-
7XSOG; 3, RRN-7XSOG; 4, RâN-7XSOG; 5, RRN2XSOG; 6, SRN-
7XSOG; 7, SRN2XSOG; 8, SRO6XSOG; 9, SâO6XSOG; 10,
RRO6XSOG; 11, RâO6XSOG. Panel C: Representative radio-
chromatogram separating the mixture of products formed in a
reaction between [14C]guanosine and (S)-p-methylstyrene oxide.
F igu r e 1. CD spectra of N2-[2-hydroxy-1-(para-substituted
phenyl)ethyl]guanines prepared by acid hydrolysis of N2-[2-
hydroxy-1-(para-substituted phenyl)ethyl]guanosines overlayed
on the CD spectra of N2-[2-hydroxy-1-(para-substituted phenyl)-
ethyl]guanines prepared by displacement of chloride from
2-chloro-6-hydroxypurine with optically active para-substituted
phenylglycinols: panel A, N2-[2-hydroxy-1-(p-methylphenyl)eth-
yl]guanines; panel B, N2-[2-hydroxy-1-(p-bromophenyl)ethyl]-
guanines.
inverted stereochemistry was 1:3 in O6-substituted prod-
ucts and between 1:5 and 1:8 in N2-substituted products.
O6-substituted products undergoing more extensive ra-
cemization than the N2-substituted products.
Discu ssion
In this study, 10 p-methyl- and 10 p-bromostyrene
oxide-guanosine markers were prepared and character-
ized spectroscopically. Stereochemical assignments of O6-
substituted diastereomeric pairs were straightforward
since each was made from an optically active diol in a
reaction that did not involve the chiral center of the
epoxide. Assignment of the N2-substituted diastereomers
was more complex since these were derived from racemic
epoxides. Assignment was achieved, however, by con-
verting these N2-substituted nucleosides, by acid hy-
drolysis, to N2-substituted guanines of known configu-
ration. Reactions of guanosine with optically active
epoxides gave optically active 7-substituted products that
were presumed to have arisen with inversion of config-
uration. With the aid of these markers, the effect of the
para substituent on configurations and relative propor-
tions of products formed in reactions of guanosine with
p-substituted styrene oxides in neutral aqueous media
could be readily determined (Table 1).
The para-substituents of the epoxides influenced the
site of ring opening by the 7-position. The p-methylsty-
rene oxide was opened by the ring nitrogen to similar
extents at the R- and â-carbons, whereas the p-bromo
substituent seemed to direct attack preferentially to the
â-carbon (almost 4-fold). All the p-substituted epoxides
were ring-opened by the exocyclic sites only at the
R-carbon. However, the para substituents did influence
the stereochemistry of products at the N2- and O6-
positions. The p-methyl substituent increased the pro-
portion of retained to inverted stereochemistry in the
products relative to the p-bromo substituent. Complete
racemization was found in the O6-substituted products
from the reactions with p-methylstyrene oxides, but a
ratio of between 1:3 and 1:2 retained-to-inverted stereo-
chemistry was found in products at the N2-position. This
contrasted with that found in reactions involving the
p-bromostyrene oxides where the ratio of retained-to-

52 Chem. Res. Toxicol., Vol. 11, No. 1, 1998
Barlow and Dipple
Ta ble 1. P r op or tion of Tota l P r od u cts F or m ed fr om Rea ction s of Op tica lly Active P a r a -Su bstitu ted Styr en e Oxid es a n d
Gu a n osin e Given a s Ra tios of Ster eoisom er s (S/R)a
epoxide
âN-7XSOGb
RN-7XSOGb
RN2XSOGb
O6(R+â)XSOGc
(R)-MeSO
<0.01/0.13
(0.025 ( 0.001)
0.01/0.46
0.16/<0.02
(9.220 ( 0.846)
0.31/0.02
0.45/0.17
(2.731 ( 0.066)
0.11/0.02
0.03/0.03
(1.03 ( 0.060)
0.04/0.03
(R)-SO*
(R)-BrSO
<0.01/0.66
(0.015 ( 0.007)
0.11/<0.01
(6.292 ( 0.519)
0.48/0.01
0.17/<0.01
0.08/<0.01
0.03/0.01
(2.604 ( 0.475)
0.04/0.03
(1.230 ( 0.060)
0.02/0.04
(22.985 ( 11.519)
<0.02/0.16
(8.260 ( 0.010)
(S)-MeSO
0.21/0.42
(0.503 ( 0.011)
0.03/0.10
(0.099 ( 0.018)
0.01/0.31
(S)-SO*
(S)-BrSO
0.66/0.00
<0.01/0.16
(0.022 ( 0.022)
<0.02/0.11
(0.173 ( 0.014)
0.01/0.03
(0.350 ( 0.010)
a
Results are given as the mean of three determinations, and the ratios (S/R) and standard deviations of the ratios are given in
parentheses. Two-tailed probability calculations were performed to determine whether the ratios of stereoisomers of the p-methyl- and
p-bromostyrene oxide products were significantly different. These ratios were not different for the âN-7XSOG and RN-7XSOG compounds
(p > 0.25); however, the ratios for the RN2XSOG (p < 0.01) and O6(R+â)XSOG (p < 0.03) compounds were different. *Data for (R)- and
(S)-styrene oxide reactions were taken from Latif et al. (18). Conversion of guanosine to products averaged ∼8% for reactions with the
b
p-methylstyrene oxide and ∼4% for reactions with the p-bromostyrene oxide. The radioactive baseline in the region of the column elution
where the 7- and N2-substituted products elute makes it difficult to convincingly demonstrate the absence or presence of minor amounts
of products. c The quantities of R- and âO6-substituted products are taken together as they ultimately arise from the RO6-substituted
compound and the resolution of peaks 10 and 11 (Figure 2) made their individual quantification difficult.
Table 1 shows that the configuration of the âN-7SOG
products did not vary with the para substituent. This is
expected as the reaction does not involve the chiral
center. As found for the â-substituted products, epoxide
ring opening at the R-carbon by the nitrogen at the
7-position gave products of predominantly one configu-
ration irrespective of the para substituent. However, in
the R-substituted products, the configuration of the
starting epoxide was inverted, consistent with a direct
displacement by an SN2 type of reaction mechanism.
Generally aliphatic epoxides open at the less substituted
carbon via a bimolecular SN2 process with inversion of
stereochemistry (31). However, as in the present case,
epoxide ring opening may also occur at the more hindered
carbon in aralkyl epoxides where a developing positive
charge can be stabilized by the aromatic substituent (32).
This charge stabilization can be increased by electron-
donating substituents and decreased by electron-with-
drawing substituents (33). In concert with this expec-
tation, the ratio of R:â 7-substituted products increased
in reactions with the p-methyl and decreased in reactions
with the p-bromo relative to reactions with the unsub-
stituted styrene oxide (Table 1).
Epoxide ring opening also occurred at the R-carbon in
reactions with the exocyclic N2- and O6-positions of
guanosine. The reaction mechanism at the three sites
on guanosine was not identical as evidenced by the
different degrees of racemization found in the products.
The degree of racemization increased in the sequence RN-
7XSOG, RN2XSOG, (R+â)O6XSOG and also with the para
substituent in the sequence -Br, -H, -Me, i.e., with
increasing ability of the para substituent to stabilize
positive charge (Table 1). The degree of racemization
ranged from essentially zero in the RN-7XSOG products
to complete racemization in the O6MeSOG products.
These findings suggest that reactions proceed by a more
ionic SN1-like mechanism at the exocyclic sites than at
the ring nitrogen. The lower degree of racemization
found in the N2- versus O6-substituted products indicates
that the exocyclic amino group requires less extensive
substrate ionization for reaction to occur compared to
reactions involving the exocyclic oxygen.
cyclic sites on guanosine. Thus, reaction occurs largely
at the R-carbon, which can support ionization through
charge delocalization in the adjacent phenyl ring and not
at the R-carbon where ionization is not favored. [Al-
though both R- and â-substituted products are found at
the O6-position, the âO6-substituted compounds arise
from rearrangement of the initially formed RO6-substi-
tuted compounds (27).]
Product distributions (Table 1) also indicate mecha-
nistic differences in reactions at the various guanosine
positions. Thus, for the p-bromostyrene oxide that would
be expected to react through the most SN2-like process,
the âN-7 products comprised 66% of the total products,
whereas with the p-methylstyrene oxide that would have
the most ionic character in its reactions forms primarily
RN2-substituted products (60-61%). The low levels of
O6-substituted product suggest that reaction at this site
requires a more energetic species, and the degree of
racemization indicates that this is of greater ionic
character than the species that reacts at the N2-position.
The ability of the epoxides in this study to ionize at
the R-carbon should increase in the order -Br < -H <
-Me, in tandem with the ability of the para substituents
to stabilize positive charge. This was reflected in the
greater proportion of R- to â-products formed in this
sequence. Since the exocyclic guanosine positions only
opened the epoxides at the R-carbon with some degree
of racemization, a more ionic intermediate is involved in
reaction at these sites. However, the degree of racem-
ization differed in the products at the various guanosine
sites indicating that the reaction mechanism was de-
pendent on both the p-substituent and the nucleophilicity
of the reaction center.
These findings extend and support previous studies
(12-18) which indicate that the ionic character of a
substrate determines its site of reaction with guanosine.
This concept rationalizes much of the literature as simple
alkyl epoxides which are not able to stabilize an ionic
charge to any great extent are found to react predomi-
nantly at ring nitrogen positions (3, 4, 34, 35). In
contrast, the polycyclic aromatic hydrocarbon dihydrodiol
epoxides, which should facilitate ionization by stabilizing
any developing charge through their multiple aromatic
ring systems, predominantly modify exocyclic amino
groups (2). Reaction at the O6-position requires ionic
The conclusion that reactions at the exocyclic sites go
through a somewhat ionic mechanism is supported by
the regiochemistry of epoxide ring opening by the exo-

Mechanisms of Guanosine Aralkylation
Chem. Res. Toxicol., Vol. 11, No. 1, 1998 53
(15) Dipple, A., Moschel, R. C., and Hudgins, W. R. (1982) Selectivity
of alkylation and aralkylation of nucleic acid components. Drug
Metab. Rev. 13, 249-268.
species of higher energy such as alkyldiazonium ions that
do not delocalize their charge (1).
(16) Moschel, R. C., Hudgins, W. R., and Dipple, A. (1979) Substituent-
induced effects on the stability of benzylated guanosines: model
systems for the factors influencing the stability of carcinogen-
modified nucleic acids. J . Org. Chem. 49, 363-372.
(17) Moschel, R. C., Hudgins, W. R., and Dipple, A. (1986) Reactivity
effects on site selectivity in nucleoside aralkylation: A model for
the factors influencing the sites of carcinogen-nucleic acid interac-
tions. J . Org. Chem. 51, 4180-4185.
(18) Latif, F., Moschel, R. C., Hemminki, K., and Dipple, A. (1988)
Styrene oxide as a stereochemical probe for the mechanism of
aralkylation at different sites of guanosine. Chem. Res. Toxicol.
1, 364-369.
Ack n ow led gm en t. Dr. C. Qian undertook some
helpful preliminary work on this project. We thank J .
Klose and M. Maguire for NMR studies, Dr. C. J . Metral
and J . Roman for MS studies, Dr. G. J anini for analysis
of the enantiopurity of the p-substituted styrene diols,
and C. Riggs for statistical analysis. We also thank Dr.
D. Whalen and Dr. R. C. Moschel for helpful discussions.
Research was sponsored by the National Cancer Institute
DHHS, under contract with ABL.
(19) Qian, C., and Dipple, A. (1995) Different mechanisms of aralky-
lation of adenosine at the 1- and N6-positions. Chem. Res. Toxicol.
8, 389-395.
(20) Sugiura, K., and Goto, M. (1981) Mutagenicities of styrene oxide
derivatives on bacterial test systems: Relationship between
mutagenic potencies and chemical reactivity. Chem.-Biol. Interact.
35, 71-91.
(21) Maltoni, C., Failla, G., and Kassapidis, G. (1979) First experi-
mental demonstration of the carcinogenic effects of styrene oxide.
Med. Lav. 5, 358-362.
(22) Lijinsky, W. (1986) Rat and mouse forestomach tumors induced
by chronic oral administration of styrene oxide. J . Natl. Cancer
Inst. 77, 471-476.
(23) Hemminki, K., and Hesso, A. (1984) Reaction products of styrene
oxide with guanosine in aqueous media. Carcinogenesis 5, 601-
607.
(24) Savela, K., Hesso, A., and Hemminki, K. (1986) Characterization
of reaction products between styrene oxide and deoxynucleosides
and DNA. Chem.-Biol. Interact. 60, 235-246.
(25) Shapiro, R., Cohen, B. I., Shiuey, S.-J ., and Maurer, H. (1969)
On the reaction of guanine with glyoxal, pyruvaldehyde, and
ketoxal, and the structure of acylguanines. A new synthesis of
N2-alkylguanines. Biochemistry 8, 238-245.
(26) Fasman, G. D. (1975) Handbook of Biochemistry and Molecular
Biology, 3rd ed., pp 419-447, CRC Press, Cleveland, OH.
(27) Moschel, R. C., Hemminki, K., and Dipple, A. (1986) Hydrolysis
and rearrangement of O6-substituted guanosine products result-
ing from the reaction of guanosine with styrene oxide. J . Org.
Chem. 51, 2952-2955.
(28) Westkaemper, R. B., and Hanzlik, R. P. (1981) Mechanistic studies
of epoxide hydrolase utilizing a continuous spectrophotometric
assay. Arch. Biochem. Biophys. 208, 195-204.
(29) Sharpless, K. B., Amberg, W., Bennani, Y. L., Crispino, G. A.,
Hartung, J ., J eong, K.-S., Kwong, H.-L., Morikawa, K., Wang, Z.-
M., Xu, D., and Zhang, X.-L. (1992) The osmium-catalysed
asymmetric dihydroxylation: A new ligand class and process
improvement. J . Org. Chem. 57, 2768-2771.
(30) Chang, H.-T., and Sharpless, K. B. (1996) A practical route to
enantiopure 1,2-amino alcohols Tetrahedron Lett. 37, 3219-3222.
(31) March, J . (1992) Advanced Organic Chemistry, 4th ed., pp 368-
369, Wiley-Interscience, New York.
(32) Laird, R. M., and Parker, R. E. (1961) The mechanism of epoxide
reaction. IV. The reactions of benzylamine with a series of m-
and p-substituted styrene oxides in ethanol. J . Am. Chem. Soc.
4277-4281.
(33) Keiyser, M. M., and Morand, P. (1980) An analysis of the factors
contributing to the regioselectivity observed in the opening of
oxiranes. Can. J . Chem. 58, 302-306.
(34) Solomon, J . J ., Singh, U. S., and Segal, A. (1993) In vitro reactions
of 2-cyanoethylene oxide with calf thymus DNA. Chem.-Biol.
Interact. 88, 115-135.
(35) Selzer, R. R., and Elfarra, A. A. (1996) Synthesis and biochemical
characterization of N1-, N2-, and N7-guanosine adducts of buta-
diene monoxide. Chem. Res. Toxicol. 9, 126-132.
Refer en ces
(1) Lawley, P. D. (1994) From fluorescence spectra to mutational
spectra, a historical overview of DNA-reactive compounds. In
DNA Adducts: Identification and Biological Significance (Hem-
minki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Seger-
ba¨ck, D., and Bartsch, H., Eds.) pp 3-22, IARC Scientific
Publications No. 125, IARC, Lyon, France.
(2) Dipple, A. (1997) DNA-reactive agents. In Comprehensive Toxicol-
ogy (Sipes, I. G., McQueen, C. A., and Gandolphi, A. J ., Eds.) pp
303-314, Elsevier Science Ltd., Oxford.
(3) Segerba¨ck, D. (1990) Reaction products in haemoglobin and DNA
after in vitro treatment with ethylene oxide and N-(2-hydroxy-
ethyl)-N-nitrosourea. Carcinogenesis 11, 307-312.
(4) Solomon, J . J ., Mukai, F., Fedyk, J ., and Segal, A. (1988) Reactions
of propylene oxide with 2′-deoxynucleosides and in vitro with calf
thymus DNA. Chem.-Biol. Interact. 67, 275-294.
(5) Segerba¨ck, D. (1994) DNA alkylation by ethylene oxide and some
monosubstituted epoxides. In DNA Adducts: Identification and
Biological Significance (Hemminki, K., Dipple, A., Shuker, D. E.
G., Kadlubar, F. F., Segerba¨ck, D., and Bartsch, H., Eds.) pp 37-
47, IARC Scientific Publications No. 125, IARC, Lyon, France.
(6) J effrey, A. M., J ennette, K. W., Blobstein, S. H., Weinstein, I. B.,
Beland, F. A., Harvey, R. G., Kasai, H., Murai, I., and Nakanishi,
K. (1976) Benzo[a]pyrene-nucleic acid derivative found in vivo:
Structure of a benzo[a]pyrene dihydrodiol epoxide-guanosine
adduct. J . Am. Chem. Soc. 98, 5714-5715.
(7) Meehan, T., Staub, K., and Calvin, M. (1977) Benzo[a]pyrene diol
epoxide covalently binds to deoxyguanosine and deoxyadenosine
in DNA. Nature 269, 725-727.
(8) Koreeda, M., Moore, P. D., Wislocki, P. G., Levin, W., Conney, A.
H., Yagi, H., and J erina, D. M. (1978) Binding of benzo[a]pyrene
7,8-diol-9,10-epoxides to DNA, RNA and protein of mouse skin
occurs with high stereoselectivity. Science 199, 778-781.
(9) Melikian, A. A., Amin, S., Hecht, S. S., Hoffmann, D., Pataki, J .,
and Harvey, R. G. (1984) Identification of the major adducts
formed by reaction with 5-methylchrysene anti-dihydrodiol-
epoxides with DNA in vitro. Cancer Res. 414, 2524-2529.
(10) Reardon, R. B., Prakash, A. S., Hilton, B. D., Roman, J . M., Pataki,
J ., Harvey, R. G., and Dipple, A. (1991) Characterisation of
5-methylchrysene-1,2-dihydrodiol-3,4-epoxide-deoxyribonucleo-
side adducts. Carcinogenesis 8, 1317-1322.
(11) Peltonen, K., Hilton, B. D., Pataki, J ., Lee, H., Harvey, R. G.,
and Dipple, A. (1991) Spectroscopic characterization of syn-5-
methylchrysene-1,2-dihydrodiol 3,4-epoxide-deoxyribonucleoside
adducts. Chem. Res. Toxicol. 4, 305-310.
(12) Brookes, P., Dipple, A., and Lawley, P. D. (1968) The preparation
and properties of some benzylated nucleosides. J . Chem. Soc.
2026-2028.
(13) Moschel, R. C., Hudgins, W. R., and Dipple, A. (1979) Selectivity
in nucleoside alkylation and aralkylation in relation to chemical
carcinogenesis. J . Org. Chem. 44, 3324-3328.
(14) Moschel, R. C., Hudgins, W. R., and Dipple, A. (1980) Aralkylation
of guanosine by the carcinogen N-nitroso-N-benzylurea. J . Org.
Chem. 45, 533-535.
TX970126I